scholarly journals Noncanonical Heme Ligands Steer Carbene Transfer Reactivity in an Artificial Metalloprotein

2021 ◽  
Author(s):  
Moritz Pott ◽  
Matthias Tinzl ◽  
Takahiro Hayashi ◽  
Yusuke Ota ◽  
Daniel Dunkelmann ◽  
...  
Keyword(s):  
Redox Potential ◽  
Stop Codon ◽  
Heme Iron ◽  
Primary Metal ◽  
Functional Roles ◽  
Trna Synthetases ◽  
Reduction Potentials ◽  
Heme Ligands ◽  
Carbene Transfer ◽  
Powerful Strategy

<a>Changing the primary metal coordination sphere is a powerful strategy for modulating metalloprotein properties. Taking advantage of this approach, we have replaced the proximal histidine ligand in myoglobin with the histidine analogues N<sub>d</sub>-methylhistidine (NMH), 5‑thiazoylalanine (5ThzA), 4-thiazoylalanine (4ThzA) and 3-(3-thienyl)alanine (3ThiA) by amber stop codon suppression using engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes. In addition to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein’s promiscuous carbene transfer activity with ethyl diazoacetate. Myoglobin variants with increased reduction potentials (NMH and 5ThzA) proved superior for cyclopropanation and N-H insertion, especially under aerobic conditions, and could even promote these reactions in the absence of reducing agent. In contrast, the variants with the lowest <i>E</i><sup>o</sup> values (4ThzA and 3ThiA) exhibit comparatively high S-H insertion activity even though the respective histidine surrogates do not coordinate the heme iron. Given the important functional roles played by histidine in many enzymes, these genetically encoded histidine analogues represent valuable tools for probing mechanism and enabling new chemistries in metalloprotein</a>s.

scholarly journals Noncanonical Heme Ligands Steer Carbene Transfer Reactivity in an Artificial Metalloprotein

2021 ◽  
Author(s):  
Moritz Pott ◽  
Matthias Tinzl ◽  
Takahiro Hayashi ◽  
Yusuke Ota ◽  
Daniel Dunkelmann ◽  
...  
Keyword(s):  
Redox Potential ◽  
Stop Codon ◽  
Heme Iron ◽  
Primary Metal ◽  
Functional Roles ◽  
Trna Synthetases ◽  
Reduction Potentials ◽  
Heme Ligands ◽  
Carbene Transfer ◽  
Powerful Strategy

<a>Changing the primary metal coordination sphere is a powerful strategy for modulating metalloprotein properties. Taking advantage of this approach, we have replaced the proximal histidine ligand in myoglobin with the histidine analogues N<sub>d</sub>-methylhistidine (NMH), 5‑thiazoylalanine (5ThzA), 4-thiazoylalanine (4ThzA) and 3-(3-thienyl)alanine (3ThiA) by amber stop codon suppression using engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes. In addition to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein’s promiscuous carbene transfer activity with ethyl diazoacetate. Myoglobin variants with increased reduction potentials (NMH and 5ThzA) proved superior for cyclopropanation and N-H insertion, especially under aerobic conditions, and could even promote these reactions in the absence of reducing agent. In contrast, the variants with the lowest <i>E</i><sup>o</sup> values (4ThzA and 3ThiA) exhibit comparatively high S-H insertion activity even though the respective histidine surrogates do not coordinate the heme iron. Given the important functional roles played by histidine in many enzymes, these genetically encoded histidine analogues represent valuable tools for probing mechanism and enabling new chemistries in metalloprotein</a>s.

Development of the Redox Probe Field Technique★

CORROSION ◽  
1958 ◽  
Vol 14 (6) ◽  
pp. 26-30 ◽  
Author(s):  
F. E. COSTANZO ◽  
R. E. McVEY
Keyword(s):  
Seasonal Variation ◽  
Redox Potential ◽  
Soil Resistance ◽  
Probe Field ◽  
Redox Probe ◽  
Field Technique ◽  
Reduction Potentials ◽  
Oxidation Reduction ◽  
Bacterial Corrosion ◽  
Profile Study

Abstract A description is given of a field technique developed to obtain oxidation-reduction potentials of soils through the use of a probe. The results of all tests with this probe indicate that redox potential can be an extremely useful tool for corrosion engineers. The instrument is rugged enough for use in the field but sensitive enough to find even small indications of bacterial corrosion. A comparison is made between redox data and the following items: (1) Pipe-to-CuSO4 profile study, (2) Four-point soil resistance survey, (3) pH readings, (4) Earth current readings, and (5) Seasonal variation. 2.4.2

scholarly journals tRNA Synthetases Are Recruited to Yeast Ribosomes by rRNA Expansion Segment 7L but Do Not Require Association for Functionality

2021 ◽  
Vol 7 (4) ◽  
pp. 73
Author(s):  
Nina Krauer ◽  
Robert Rauscher ◽  
Norbert Polacek
Keyword(s):  
Ribosome Biogenesis ◽  
Protein Biosynthesis ◽  
Ribosomal Subunit ◽  
Trna Synthetase ◽  
Translation Efficiency ◽  
Functional Roles ◽  
Trna Synthetases ◽  
Yeast Ribosomes ◽  
Ribosome Association

Protein biosynthesis is essential for any organism, yet how this process is regulated is not fully understood at the molecular level. During evolution, ribosomal RNA expanded in specific regions, referred to as rRNA expansion segments (ES). First functional roles of these expansions have only recently been discovered. Here we address the role of ES7La located in the large ribosomal subunit for factor recruitment to the yeast ribosome and the potential consequences for translation. Truncation of ES7La has only minor effects on ribosome biogenesis, translation efficiency and cell doubling. Using yeast rRNA deletion strains coupled with ribosome-specific mass spectrometry we analyzed the interactome of ribosomes lacking ES7La. Three aminoacyl-tRNA synthetases showed reduced ribosome association. Synthetase activities however remained unaltered suggesting that the pool of aminoacylated tRNAs is unaffected by the ES deletion. These results demonstrated that aminoacylation activities of tRNA synthetases per se do not rely on ribosome association. These findings suggest a role of ribosome-associated aminoacyl-tRNA synthetase beyond their core enzymatic functions.

scholarly journals A heme-binding domain controls regulation of ATP-dependent potassium channels

2016 ◽  
Vol 113 (14) ◽  
pp. 3785-3790 ◽  
Author(s):  
Mark J. Burton ◽  
Sofia M. Kapetanaki ◽  
Tatyana Chernova ◽  
Andrew G. Jamieson ◽  
Pierre Dorlet ◽  
...  
Keyword(s):  
Metal Ion ◽  
Single Channel ◽  
Heme Iron ◽  
Receptor Subunit ◽  
Heme Binding ◽  
Prosthetic Group ◽  
Sulphonylurea Receptor ◽  
Spectroscopic Analyses ◽  
Heme Ligands

Heme iron has many and varied roles in biology. Most commonly it binds as a prosthetic group to proteins, and it has been widely supposed and amply demonstrated that subtle variations in the protein structure around the heme, including the heme ligands, are used to control the reactivity of the metal ion. However, the role of heme in biology now appears to also include a regulatory responsibility in the cell; this includes regulation of ion channel function. In this work, we show that cardiac KATP channels are regulated by heme. We identify a cytoplasmic heme-binding CXXHX16H motif on the sulphonylurea receptor subunit of the channel, and mutagenesis together with quantitative and spectroscopic analyses of heme-binding and single channel experiments identified Cys628 and His648 as important for heme binding. We discuss the wider implications of these findings and we use the information to present hypotheses for mechanisms of heme-dependent regulation across other ion channels.

Noncanonical heme ligands steer carbene transfer reactivity in an artificial metalloenzyme

2021 ◽  
Author(s):  
Moritz Pott ◽  
Matthias Tinzl ◽  
Takahiro Hayashi ◽  
Yusuke Ota ◽  
Daniel Dunkelmann ◽  
...  
Keyword(s):  
Heme Ligands ◽  
Carbene Transfer ◽  
Artificial Metalloenzyme

scholarly journals The Interaction of Covalently Bound Heme with the CytochromecMaturation Protein CcmE

2004 ◽  
Vol 279 (50) ◽  
pp. 51981-51988 ◽  
Author(s):  
Takeshi Uchida ◽  
Julie M. Stevens ◽  
Oliver Daltrop ◽  
Edgar M. Harvat ◽  
Lin Hong ◽  
...  
Keyword(s):  
Plant Mitochondria ◽  
Heme Iron ◽  
Histidine Residue ◽  
Axial Ligands ◽  
Heme Ligands ◽  
Essential Function ◽  
Resonance Raman Spectra ◽  
Vinyl Group ◽  
Covalently Bound

The heme chaperone CcmE is a novel protein that binds heme covalently via a histidine residue as part of its essential function in the process of cytochromecbiogenesis in many bacteria as well as plant mitochondria. In the continued absence of a structure of the holoform of CcmE, identification of the heme ligands is an important step in understanding the molecular function of this protein and the role of covalent heme binding to CcmE during the maturation ofc-type cytochromes. In this work, we present spectroscopic data that provide insight into the ligation of the heme iron in the soluble domain of CcmE fromEscherichia coli. Resonance Raman spectra demonstrated that one of the heme axial ligands is a histidine residue and that the other is likely to be Tyr134. In addition, the properties of the heme resonances of the holo-protein as compared with those of a form of CcmE with non-covalently bound heme provide evidence for the modification of one of the heme vinyl side chains by the protein, most likely the 2-vinyl group.

Probing electrostatic interactions in cytochrome c using site-directed chemical modification

2002 ◽  
Vol 80 (2) ◽  
pp. 197-203 ◽  
Author(s):  
Christian Blouin ◽  
J Guy Guillemette ◽  
Carmichael JA Wallace
Keyword(s):  
Cytochrome C ◽  
Chemical Modification ◽  
Redox Potential ◽  
Protein Interactions ◽  
Electrostatic Interactions ◽  
Heme Iron ◽  
Dielectric Constants ◽  
Protein Protein Interactions ◽  
Potential Control ◽  
Electrostatic Probe

This communication reports the generation of an electrostatic probe using chemical modification of methionine side chains. The alkylation of methionine by iodoacetamide was achieved in a set of Saccharomyces cerevisiae iso-1-cytochrome c mutants, introducing the nontitratable, nondelocalized positive charge of a carboxyamidomethylmethionine sulfonium (CAMMS) ion at five surface and one buried site in the protein. Changes in redox potential and its variation with temperature were used to calculate microscopic effective dielectric constants operating between the probe and the heme iron. Dielectric constants (ε) derived from ΔΔG values were not useful due to entropic effects, but εΔΔH gave results that supported the theory. The effect on biological activity of surface derivatization was interpreted in terms of protein–protein interactions. The introduction of an electrostatic probe in cytochrome c often resulted in marked effects on activity with one of two physiological partners: cytochrome c reductase, especially if introduced at position 65, and cytochrome c oxidase, if at position 28.Key words: protein engineering, chemical modification, cytochrome c, electron transport, protein electrostatics, redox potential control.

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